The present invention relates to methods and compositions for regulating gene expression in a cell. In particular, the invention provides multi-state genetic oscillator constructs in which expression of a gene of interest changes periodically in the absence of extraneous agents.
Many areas of biotechnology involve regulating the expression of one or more genes of interest by applying an external agent. Typical approaches for regulating gene expression involve natural or engineered transcription factors that activate or inhibit expression of a specific gene in response to a chemical agent [Gossen and Bujard, Proc. Natl. Acad. Sci. USA, 89:5547, 1992; Rivera, et al., Nat. Med., 2:1028, 1996; Yao and Evans, Proc. Natl. Acad. Sci. USA, 93:3346, 1996; Wang, et al., Proc. Natl. Acad. Sci. USA, 91:8180, 1994]. Transcription factors often are introduced into a cell using DNA constructs that express a transcription factor and a gene of interest. Stable activation or inhibition of a transcription factor typically requires that a chemical agent or other stimulus be applied. However, when an oscillating level of expression is desired, it is often problematic to repeatedly apply and remove a stimulus in order to obtain the oscillation. Therefore, there is an ongoing need in the art for self-contained methods and compositions that permit oscillating levels of gene expression in the absence of an external stimulus.
The invention provides a recombinant multi-state genetic oscillator that can transition the expression of one or more genes of interest from stable xe2x80x9conxe2x80x9d to stable xe2x80x9coffxe2x80x9d states or vice versa in a regular and periodic manner. According to the invention, the expression of one or more genes in the multi-state oscillator are changed from a first expression state to a second expression state by the presence of a preselected amount i.e., an amount of an activating agent equal to or greater than a specific concentration (threshold concentration). When the concentration of the activating agent drops below the threshold concentration, the oscillator returns back to its first expression state. An important feature of the invention is that the concentration of the activating agent is controlled by the expression of one or more regulatory genes in the oscillator switch. The expression of these genes, in turn, is regulated by the state of the multi-state oscillator. Thus a feedback loop is created which causes the oscillator to transition repeatedly and continuously between its two expression states. These repeated transitions produce a periodic change in concentration of the activating agent, the expression of the regulatory gene or genes, and the expression of the gene or genes of interest.
The multi-state oscillator of the invention is characterized as having two alternative expression states. In a first state, a first, inducible, promoter is substantially inactive and a second, constitutive, promoter is active. In a second state, the inducible promoter is active and the constitutive promoter is substantially inactive. Therefore, in the first state, genes that are transcribed from the constitutive promoter are expressed, and in the second state, genes that are transcribed from the inducible promoter are expressed. According to the invention, the activating agent causes a transition from the first expression state to the second expression state when it reaches a preselected or threshold concentration. Once the concentration of activating agent drops below this level, the switch returns back to the first expression state. An important feature of the invention is that the cyclical nature of the switching is controlled by periodic increases and decreases in the concentration of the activating agent. Furthermore, the periodic changes in the concentration of the activating agent are controlled by one or more regulatory genes in the oscillator. The expression of these regulatory genes, in turn, is controlled by the expression state of the oscillator. Thus a feedback loop is created which causes the oscillator to transition periodically between its expression states. By adjusting the rates and/or levels of expression of the activating agent, it is possible to produce an oscillator switch that has a desired periodicity, i.e., the oscillator cycles between its two different expression states and the switch is in one of its different expression states for a desired period of time. Preferably, the transition from the first expression state to the second expression state does not occur in the absence of the threshold concentration of the activating agent. More preferably, the transition from the second expression state to the first expression state does not occur in the presence of a concentration of activating agent equal to or greater than the threshold concentration.
With reference to FIG. 1, the components of a multi-state oscillator of the invention include a first regulatory gene (R1) that is expressed from a first, inducible promoter (P1), and a second regulatory gene (R2) that is expressed from a second, constitutive promoter (P2). A product of the first regulatory gene inhibits or reduces expression of the second regulatory gene, and a product of the second regulatory gene inhibits or reduces expression of the first regulatory gene. According to preferred embodiments, either regulatory gene can be expressed, but both preferably are not maximally expressed simultaneously. In a first expression state, the constitutive promoter (P2) is active, and the inducible promoter (P1) is substantially inactive (due to the absence of a sufficient amount of an activating agent and the inhibitory effect of the second regulatory gene product).
The presence of an activating agent (X) activates the inducible promoter (P1) to transition the switch of the invention to a second expression state in which the inducible promoter (P1) is active and the constitutive promoter (P2) is substantially inactive. However, an important feature of the invention is that a minimal threshold amount of the activating agent is required to cause the transition to the second expression state. In the presence of a threshold concentration of activating agent (X), expression from the inducible promoter (P1) is sufficient to inhibit expression from the constitutive promoter (P2). While the concentration of the activating agent is maintained above the threshold concentration, the second expression state is maintained. When the concentration of the activating agent falls below the threshold concentration, the first expression state is reestablished. Furthermore, the cycling of the oscillator is caused by the periodic changes in concentration of the activating agent, which in turn is regulated by periodic changes in the expression of one or more regulatory genes disposed within the oscillator (dashed lines in FIG. 1), which in turn are regulated by the expression state of the oscillator. Accordingly, the switch of the invention may transition periodically between its two expression states in the absence of exogenously added agents.
In a preferred embodiment, the activating agent which acts to induce expression from the inducible promoter of the oscillator is a protein. Expression of the activating protein preferably is under the control of an additional promoter. The expression and increase in concentration of the activating protein is coupled with the oscillator such that as the inducible promoter of the oscillator is turned xe2x80x9conxe2x80x9d, expression of the activating agent is turned xe2x80x9coffxe2x80x9d. Similarly, as the inducible promoter of the oscillator is turned xe2x80x9coffxe2x80x9d, expression of the activating agent is turned xe2x80x9conxe2x80x9d. This results in a switch which transitions between its two expression states in a periodic manner.
According to the invention, the inhibitory effects of the regulatory gene products expressed from the inducible and constitutive promoters is dependent on both the expression level of the gene product and the inherent inhibitory properties of the respective gene products. Expression level is a function of promoter strength, RNA stability, translational efficiency (if the gene product is a protein), post translational modification and protein stability (if the gene product is a protein). Accordingly, an activating agent that increases the expression level of the inducible regulatory gene product by increasing transcription, translation, RNA stability, post translational modification, protein stability, or a combination of the above, can cause a switch in expression states once it reaches a threshold concentration.
According to a preferred embodiment of the invention, the multi-state oscillator switch of the invention is a DNA construct comprising a network of genes such that expression of a gene of interest may be increased or decreased with regular periodicity. According to the invention, one or more genes of interest can be linked to the inducible and/or constitutive promoters of the multi-state oscillator switch of the invention. Alternatively, the gene or genes of interest may be transcribed from a separate promoter that is identical to one of the promoters in the switch or is regulated in the same way as one of the promoters of the switch.